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Single-cell mitochondrial genotypoing of exonuclease-deficient DNA polymerase γ (POLGD274A) knock-in HEK293 cell lines
No full textExonuclease-deficient DNA polymerase γ (POLGD274A) lead to an elevated mutational rate and hypermutated mitochondrial genomes. Here, leverage mitochondrial single-cell ATAC-seq (mtscATAC-seq) to estimate genome-wide mtDNA mutational burden in HEK293 cell lines with POLGD274A knock-in. Our method sensitively detects up to a a median of 472 variants per cell, corresponding to a 50-fold increase comared to control, with a predominance of replication-driven C>T transitions
Amyloid-beta (1-40) peptide is associated with systemic metabolic health
Full text linkBACKGROUND: Amyloid-beta 1–40 peptide (Aβ40) has recently emerged as a blood-based biomarker of cardiovascular disease (CVD). However, whether plasma levels of Aβ40 are associated with metabolic traits in humans without established CVD remains poorly understood. METHODS: Aβ40 was measured in plasma by ELISA and metabolic traits (waist circumference, fasting triglycerides, fasting HDL cholesterol and fasting glucose) were determined in a general population (n = 449) of individuals who did not have clinically overt CVD. Triglyceride-glucose index (TyG) was used to calculate the risk for insulin resistance. BARD score was used to calculate the risk for metabolic liver disease. RESULTS: Aβ40 levels were associated with the presence of metabolic syndrome (OR: 1.41 95% CI: 1.13-1.76, p = .003), and with higher odds for increasing incidence of metabolic syndrome components, characterized by decreased HDL-C levels (OR: 1.31 95% CI: 1.03-1.58, p = .017) and increased triglyceride levels (OR: 1.30 95% CI: 1.04-1.57, p = .033) after adjustment for traditional cardiovascular risk factors. Further, Aβ40 levels were associated with increased odds for TyG (OR: 1.26 95% CI: 1.03-1.57, p = .042) and increased odds for the presence of diabetes mellitus (OR: 1.35 95% CI: 1.04-1.76, p = .018) after adjustment for age and sex, smoking status, hypertension and dyslipidemia. Increased Aβ40 levels were associated with increased odds for BARD score ≥2 (OR: 1.41 95% CI: 1.04-2.04, p = .045) after adjustment for traditional cardiovascular risk factors. CONCLUSION: Our findings suggest that Aβ40 peptide is associated with metabolic traits and risk for metabolic disease. Future longitudinal studies are warranted to determine the prognostic value of Aβ40 for the development and progression of metabolic diseases
DNA methylation analysis of NOTCH1 variants reveals the first episignature for non-syndromic congenital heart defects
Full text linkBACKGROUND: Congenital heart defects (CHDs) are the most common malformation amongst newborns, with a prevalence of approximately 0.8–2%. The etiology of CHD is highly complex and can be linked to genetic and nongenetic factors. The molecular basis remains partially unclear, and only a minority of patients can be assigned to clear monogenic causes. METHODS: Here we analyzed a cohort of 3907 CHD cases and population-matched controls using exome sequencing. In addition, we employed epigenetic profiling on a subset of cases that harbored rare NOTCH1 variants. RESULTS: We identified 24 pathogenic or likely pathogenic single nucleotide variants (SNVs) in NOTCH1 in our exome cohort, as well as a further 15 variants of uncertain significance (VUS) likely to have a deleterious effect. Although the cardiac phenotypes showed some heterogeneity, non-syndromic Tetralogy of Fallot (ToF) and related malformations were the most frequent finding in 56% (22/39). In particular, missense variants altering cysteine residues involved in forming disulfide bridges were identified, specifically in TOF patients. Altogether, NOTCH1-haploinsufficiency represented the most common monogenic cause in our cohort and accounted for an estimated 1% of CHD cases. Combined with additional cases assembled through collaborations, we present 67 individuals with ultrarare variants affecting NOTCH1. This prominent role of NOTCH1 calls for an accurate and accessible evaluation of variants. To this end we explored DNA methylation testing and successfully established a NOTCH1-specific episignature. This signature also displays a robust specificity in relation to 99 other episignatures. Taken together, we found that truncating, splice-altering, as well as missense NOTCH1 variants, can generate a distinct DNAm episignature. CONCLUSIONS: We identified that NOTCH1-haploinsufficiency variants represented the most common monogenic cause in our cohort and accounted for an estimated 1 % of CHD cases. Furthermore, we conclude that methylation profiling can contribute to (NOTCH1) variant interpretation and improve the diagnostic management of CHD patients. Lastly, we established a NOTCH1-specific episignature, which represents the first non-syndromic signature, significantly extending the scope of patients that can benefit from methylation analysis
Comparison of different decellularization protocols for porcine centrum tendineum diaphragmatis and diaphragmatic muscle - a base for effective recellularization
Full text linkDiaphragmatic dysfunction results from a variety of diseases or post-surgical conditions, leading to impaired lung function and high morbidity and mortality. Current repair materials are limited by poor biocompatibility, functional incompatibility and immune reactions. Tissue engineering via decellularization offers a promising approach by preserving the extracellular matrix while reducing host immune response. However, most studies have focused on rodent models. This study evaluates three decellularization protocols using porcine tissues to increase clinical relevance and optimize diaphragm repair strategies. We compared detergentenzymatic treatment (DET) adapted from murine diaphragm developed by Andreas et al. (P1) and two decelularization protocols for larger mammalian diaphragm tissues published by Barbon et al. (P2) for human diaphragm and Deeken et al. (P3) for porcine tendinous diaphragm. Decellularized samples were analyzed using histological analysis, SEM, DNA and GAG quantification and proteomic analysis. DNA content was reduced in decellularized tissues significantly between native and decellularized tissues (native: 990.30 ng/mg (IQR = 556.20 ); P1: 31.92 ng/mg (IQR = 40.38), P2: 32.38 ng/mg (IQR = 20.83), P3: 106.40 ng/mg (IQR = 811.32). Proteomics revealed 4,640 conserved proteins (5.41% classified as matrisomal proteins). The protocols showed a 55.4% concordance of the preserved matrisomal fraction (n=139). The highest protein preservation was achieved by P3, followed by P2 and P1. The P1 and P2 protocols are effective in preserving the extracellular matrix while removing cellular components, with no clear preference. Within our laboratory setting, P3 showed decellularization, but did not reach current decellularization standards. This study advances the preparation for clinical translation of a decellularized porcine diaphragm
Health status, health behavior and perceived stress of nursing staff in Germany: a scoping review
Full text linkOBJECTIVE: In view of increasing work-related burdens resulting from staff shortages, demographic changes, and high physical and psychological demands, there is a growing need for an understanding of the health status of nursing staff in Germany. The aim of this review is to consolidate existing knowledge on nurses’ health, health behaviors, and subjective stress perceptions to highlight existing research gaps, and to provide impetus for the development of future health-promoting interventions. METHODS: To analyze the research field, a scoping review was conducted following the JBI methodology. The systematic literature search was carried out using CINAHL, PubMed, and CareLit- databases and was supplemented by searches of the preprint servers OpenGrey and MedRxiv. In addition, a targeted supplementary search for relevant publications was also conducted on selected pertinent websites. RESULTS: A total of 11,006 titles and abstracts were screened, of which 150 full texts were reviewed, resulting in the inclusion of 90 studies. The literature predominantly focused on nurses’ mental health. Physical health and health-related behaviors were examined less frequently. Results consistently indicate a high burden of morbidity and substantial work-related stress, which have significant implications for individual well-being, professional performance, and long-term retention in the nursing profession. These challenges have been further intensified by the COVID-19 pandemic. Protective factors such as team cohesion and recognition have emerged repeatedly, highlighting the importance of supportive work environments. Although some interventions have demonstrated short-term improvements in mental health outcomes, robust evidence of long-term effects and physical health promotion remains limited. CONCLUSIONS: Nursing staff are exposed to a wide range of health risks and high work-related burdens. Despite a broad body of research, substantial gaps remain - particularly regarding health behaviors and physical health. Future research requires longitudinal, comparative studies, and a structured, nursing-specific health monitoring system. In practice, comprehensive strategies that combine individual-level interventions with structural improvements in the work environment are needed. REVIEW REGISTRATION: Open Science Framework https://doi.org/10.17605/OSF.IO/HX9ZM. CLINICAL TRIAL NUMBER: Not applicable
Ketogene Ernährung [Ketogenic diet]
No full textVon den drei Makronährstoffen in unserer Ernährung sind nur Kohlenhydrate nicht essenziell. Bei deutlicher Einschränkung der Kohlenhydratzufuhr wird ein physiologischer Prozess in Gang gesetzt, der zur Bildung von Ketonkörpern aus Fetten führt, die sogenannte Ketogenese. Im Gegensatz zum Fasten geschieht dies im Rahmen einer ketogenen Diät (KD) nicht ausschließlich aus körpereigenen Fettreserven, sondern auch aus zugeführten Nahrungsfetten (Beispielmahlzeit in . Abb. 12.1). Bereits vor etwa 100 Jahren wurde erkannt, dass eine KD wie das Fasten eine Ketose induziert, aber langfristiger, z. B. zur Therapie von therapierefraktärer Epilepsie und Diabetes Typ 2, eingesetzt werden kann. In diesem Beitrag lesen Sie über die Entstehung der KD und was sie ausmacht, die vermuteten Wirkweisen, Formen und aktuellen Anwendungsgebiete der KD, Aspekte der praktischen Umsetzung in der Ernährungstherapie, Messung und Beurteilung von KetonWerten, sowie Compliance, Risiken, Kontraindikationen und kritische Aspekte
Lipid-facilitated opening of the ADAM10 sheddase revealed by enhanced sampling simulations
Full text linkADAM10 is a crucial membrane-bound metalloprotease that regulates cellular physiology by cleaving and releasing membrane- anchored proteins, including adhesion molecules and growth factor precursors, thereby modulating cell signaling, adhesion, and migration. Despite its central role, its activation mechanisms are not fully understood. Here, we model how phosphatidylserine (PS) exposure during apoptosis triggers ADAM10 activation. We confirm that PS externalization is associated with ADAM10- mediated CD43 shedding from the surface of T cells. Intriguingly, ADAM10 activation correlated with loss of ADAM10 monoclonal antibody binding, suggesting a PS-induced conformational change that alters epitope accessibility. To explore this lipid-mediated conformational change of ADAM10, we employed molecular dynamics simulations to map its conformational landscape. Our simulations revealed that in the absence of PS, ADAM10 samples predominantly closed and intermediate states. By contrast, the presence of PS destabilizes the closed conformation, thereby favoring open states. We provide a mechanistic explanation for this PS-induced conformational change, which drives ADAM10 activation and loss of mAb binding through conformational change. These findings offer new insights into the lipid-mediated regulation of ADAM10 and its conformational dynamics
Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR
No full textTranscriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies
Der Mensch ist, was er isst – wie Essen unsere seelische Gesundheit prägt [You are what you eat - How food shapes our mental health]
No full text„Der Mensch ist, was er isst“ – diesen Gedanken formulierte der Philosoph Ludwig Feuerbach bereits im Jahr 1850. Schon die Römer wussten: Mens sana in corpore sano – ein gesunder Geist wohnt in einem gesunden Körper. Doch gilt das auch für die Psyche? Wird man anfälliger für Depressionen oder Angststörungen, wenn man sich täglich von Fast Food ernährt? Und warum bleiben manche Menschen trotz ungesunder Lebensweise psychisch stabil, während andere schnell aus dem seelischen Gleichgewicht geraten
Microstructure-informed deep learning improves thalamic atrophy segmentation and clinical associations in multiple sclerosis and related neuroimmunological diseases
Full text linkThalamic atrophy is a sensitive imaging marker of neurodegeneration in multiple sclerosis (MS) and related disorders, though thalamus segmentation remains method-dependent. Quantitative magnetic resonance imaging (qMRI) may enhance thalamic boundary contrast, particularly in the context of deep learning. We benchmarked thalamic segmentations from two atlas-constrained algorithms, FreeSurfer and FIRST, and two deep learning algorithms, DBSegment and MindGlide (an MS-trained model), against ground truth (GT) labels, tested whether quantitative R1 maps improve performance, and evaluated clinical validity cross-sectionally and longitudinally. We generated thalamus masks using each algorithm from T1-weighted data in a single-scanner cohort (baseline n = 321; 1-year follow-up n = 234) including patients with MS/related disorders and healthy controls. Using MindGlide, we also produced FLAIR- and R1-based masks and ensembles. Manual GT labels were obtained for 50 MS patients using T1w and FLAIR scans. For voxel-wise GT agreement, DBSegment yielded the highest Dice-similarity coefficient; atlas-constrained methods showed the highest sensitivity but lowest precision, while MindGlide balanced both. Volumetrically, MindGlide showed the most accurate estimates; DBSegment and FreeSurfer showed proportional bias, and both atlas-constrained methods overestimated thalamic volumes. Adding R1 input to MindGlide produced modest or no gains in GT agreement. Additionally, MindGlide volumes were most consistently associated with disability and cognitive scores cross-sectionally, and longitudinally showed the largest effects between thalamic volume change and EDSS worsening. Incorporating R1 maps offered no cross-sectional benefit but strengthened longitudinal associations. Higher-resolution qMRI and multi-contrast deep learning architectures may further enhance thalamic segmentation and monitoring in neuroinflammatory diseases